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Maintenance of Akt activity is required for the aggressive, glucose-avid cancer cell phenotype. The myrAkt-3-D1 cell line was derived from a leukemic myrAkt-induced mouse by culturing cells in the presence of G418 drug selection. The myrAkt-3-D1 cells were expanded in vitro and then transferred to naïve nude mice. A, mice receiving no cells, Bcl-xL-expressing cells, or myrAkt-3-D1 cells with or without doxycycline exposure were monitored for overall survival. Mice were sacrificed when visibly ill. P (for survival of recipients of myrAkt-3-D1 cells with versus without doxycycline treatment, P < 0.05) was determined by Student’s t test. B, mice receiving myrAkt-3-D1 cells with or without doxycycline treatment were imaged by 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) 14–21 days after transfer of cells. Mice were i.v. administered 30 μCi of FDG, and 60 min later, PET images were acquired. Representative images are shown.

Akt stimulates aerobic glycolysis. A, vector control or myrAkt-3 cells in the absence or presence of doxycycline were assayed for whole cell NAD(P)H fluorescence. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone, potassium cyanide (KCN), and iodoacetic acid (IAA) were added at 200, 400, 600, and 1000 s, respectively. The traces from a representative experiment are shown. B, IAA-sensitive NAD(P)H was calculated as the change in fluorescence in response to IAA treatment. C and D, vector control or myrAkt–3 cells in the absence or presence of doxycycline were assayed for rates of glycolysis and oxygen consumption after 48 h in culture. C, glycolytic rate was measured in cells incubated with a 5-3H-glucose tracer by the production of [3H]2O by enolase. D, cellular oxygen consumption was measured in an airtight chamber with an oxygen electrode. Values shown are the means of three independent experiments. Error bars represent SDs.

Leukemic Akt-expressing cells demonstrate increased rates of glucose metabolism and require continuous glucose supply for a survival advantage. A and B, cell lines obtained from three leukemic mice (myrAkt-3-D1, myrAkt-3-D2, and myrAkt-3-D3) were cultured without passage in the absence or presence of doxycycline. Cells were seeded at 5 × 104 cells/ml. A, cell concentration was determined with a Coulter Z2 particle analyzer. Cell viability was determined by propidium iodide exclusion. B, glucose and lactate were quantified using colorimetric reactions. Glucose and lactate concentrations were normalized to cell number in the presence of doxycycline. Results were averaged for the three lines. Values shown are the means of three independent experiments. Error bars represent standard errors of the mean. C, the myrAkt-3-D1-leukemic cells were cultured without passage in the absence (left) or presence (right) of doxycycline. Cells were seeded at 5 × 104 cells/ml. Cultures were either supplemented or not after 4 days with 3.5 mm glucose. Cell viability was determined by propidium iodide exclusion. Values shown are the means of three independent experiments. Error bars represent SDs.

LN18 cells take up glucose and secrete lactate to a greater extent than LN229 cells, and LN18 cells are dependent on glucose for viability. LN18 or LN229 glioblastoma cells were plated at 105/ml and cultured for 4 days in the absence of serum. Cells were counted daily (A). Aliquots of medium were removed at each time point and tested for glucose (B) and lactate (C). D, viability was tested by propidium iodide exclusion. Data are representative of three independent experiments. E, Akt activity was evaluated by Western blot with antibody against phospho-Akt Ser473 (P-Akt) in cells deprived of serum.

The phosphatidylinositide-3-kinase inhibitor LY294002 (LY) causes dephosphorylation of Akt in LN18 cells and inhibits glucose uptake and lactate production. A, Akt phosphorylation in LN18 cells was assayed by Western blot for phospho-Ser473 in the absence or presence of LY. B and C, cells were cultured as in Fig. 4
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in the presence or absence of LY and glucose (B) and lactate (C) levels were assayed every day.

Expression of myrAkt in LN229 cells drives glucose uptake and lactate production. A, LN229 cells were transfected with pSFFV plasmid alone or pSFFV containing a myrAkt construct. Levels of phosphorylated (P-Akt) and total Akt were determined by Western blot analysis. LN229 represents the parental cell line; myrAkt-1 and myrAkt-2 represent two independent clones stably transfected with myrAkt; vec-1 and vec-2 are two independent clones stably transfected with the empty vector. Cells were cultured in the presence or absence of LY294002 (LY) for 8 h before protein isolation. B, control and transfected cells were cultured as in Fig. 4
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, and cell numbers were determined every day. Glucose (C) and lactate (D) levels were assayed at each time point of the experiment presented in B. E, cells were cultured in the absence of glucose, and viability was determined by propidium iodide exclusion. Data are representative of three independent experiments.